Chin Bull Bot ›› 2018, Vol. 53 ›› Issue (5): 643-652.doi: 10.11983/CBB17173

• EXPERIMENTAL COMMUNICATIONS • Previous Articles     Next Articles

Mass Propagation and Genetic Stability of Bergenia Species

Lü Xiuli1,2,3†, Zhang Qun1†, Chen Xiangbo1, Li Pujin1, Wu Wei4, Guan Yuan5,6,*   

  1. 1Shanghai Academy of Landscape Architecture Science and Planning, Shanghai 200232, China
    2Shanghai Engineering Research Center of Landscaping on Challenging Urban Sites, Shanghai 200232, China
    3National Forest Genetic Resources Platform-Shanghai Sub-platform, Shanghai 200232, China
    4Shanghai Botanical Garden, Shanghai 200232, China
    5Forestry and Fruit Tree Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China
    6Shanghai Key Laboratory of Protected Horticultural Technology, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China
  • Received:2017-09-10 Accepted:2018-03-13 Online:2018-11-29 Published:2018-09-01
  • Contact: Guan Yuan
  • About author:

    † These authors contributed equally to this paper

Abstract:

According to commercial use, market demand and survival of wild resources, Bergenia crassifolia, B. scopulosa, B. purpurascens were selected for tissue culture in vitro and standardized propagation. ISSR markers were used to analyze the genetic stability of tissue culture plantlets. The optimal multiplication medium was MS medium supplemented with 0.01 mg·L-1 NAA, 0.5 mg·L-1 6-BA, and 2.0 mg·L-1 Vc with shoot tips used as explants. The multiplication coefficients were 3.10, 2.50 and 2.10 for the three species, respectively. The suitable rooting culture medium was 1/2MS medium with 1.0 mg·L-1 IBA and 2.0 mg·L-1 Vc, and the rooting rates for the three Bergenia species were 85%, 80%, and 75%, respectively. The rooted plants were transplanted in a mixed medium of humus, yellow sand, and perlite with volume ratio 2:1:1; the survival rates of transplanted plantlets were 90%, 85% and 80%, respectively. Using this method of rapid propagation, three Bergenia species propagated 200 000, 20 000, and 10 000 plantlets, respectively. ISSR molecular marker detection results showed that genetic variation was greater for regenerated plantlets of B. purpurascens than the mother plant and was lower for regenerated plantlets of B. scopulosa than the mother plant. After 20 generations of regeneration, the plantlets of the 3 Bergenia species showed genetic variation. The average genetic variation rate of B. scopulosa and B. purpurascens increased with increasing subculture times, but the average genetic variation rate of B. crassifolia after the increase in number of generations changed irregularly.

Key words: Bergenia crassifolia, B. scopulosa, B. purpurascens, genetic stability, mass propagation

Table 1

The sequence of ISSR primers"

Name of
primers
Sequence of primers (5'-3') Annealing
temperature (°C)
UBC815 CTCTCTCTCTCTCTCTG 50.6
UBC845 CTCTCTCTCTCTCTCTRG 51.8
UBC852 TCTCTCTCTCTCTCTCRA 51.6
UBC853 TCTCTCTCTCTCTCTCRT 50.9
UBC854 TCTCTCTCTCTCTCTCRG 53.4

Table 2

Effects of different concentrations of VC on the ste- rile seedlings"

VC
(mg·L-1)
Aseptic seedling (individual) Sterile seedling
rate (%)
Browning
condition
0.1 0±0 e 0±0 e Browning
0.2 0±0 e 0±0 e Browning
0.5 8±2 c 40±10 c No browning
1.0 13±1 b 65±5 b No browning
2.0 15±0 a 75±0 a No browning
4.0 14±1 ab 70±5 ab No browning
5.0 13±0 b 65±0 b No browning
6.0 4±0 d 20±0 d No browning
8.0 0±0 e 0±0 e Death

Table 3

Effects of different explants on the sterile seedlings"

Explant Inoculation No. (individual) Septic seedling No. (individual) Sterile seedling rate (%)
Top bud of mother plant 30 4±1 a 13.33±0.03 a
Shoot apical bud of ramet 30 25±2 b 83.33±0.07 b

Table 4

The effects of different hormone and hormone concentrations on the germination of bud of 3 Bergenia species"

Concentration (mg·L-1) No. of germination
(individual)
Germination rate
(%)
Other condition
6-BA NAA
0.01 0.01 5±0 d 25±0 d No callus, undifferentiated
0.01 0.02 12±1 b 60±5 b No callus, undifferentiated
0.01 0.04 10±0 c 50±0 c Callus, undifferentiated
0.01 0.08 10±1 c 50±5 c Callus, undifferentiated
0.01 0.1 10±0 c 50±0 c Callus, undifferentiated
0.01 0.2 10±1 c 50±5 c Callus, undifferentiated
0.01 0.5 10±2 c 50±10 c Callus, undifferentiated
0.02 0.02 15±1 a 75±5 a No callus, undifferentiated
0.05 0.02 15±2 a 75±10 a No callus, undifferentiated
0.1 0.02 15±0 a 75±0 a No callus, undifferentiated

Figure 1

Tissue culture and rapid propagation of 3 Bergenia species(A)-(C) Multiplication culture of B. crassifolia (A), B. scopulosa (B) and B. purpurascens (C); (D)-(F) Rooting culture of B. crassifolia (D), B. scopulosa (E) and B. purpurascens (F); (G)-(I) Transplanting of B. crassifolia (G), B. scopulosa (H) and B. purpurascens (I)"

Table 5

The effects of different hormone and hormone concentrations on shoot multiplication of 3 Bergenia species"

Concentration (mg·L-1) No. of multiplication bud (individual) Multiplication coefficien Browning condition
6-BA NAA VC A B C A B C A B C
0.1 0.01 24±1 g 29±2 e 23±0 de 1.20±0.03 g 1.45±0.07 e 1.15±0 de + - -
0.1 0.01 2.0 33±2 f 27±1 e 22±2 e 1.65±0.07 f 1.35±0.03 e 1.10±0 e - - -
0.25 0.01 31±1 f 33±2 d 25±1 d 1.55±0.03 f 1.65±0.07 d 1.25±0.03 d + - -
0.25 0.01 2.0 45±2 d 36±1 c 31±2 c 2.25±0.07 d 1.80±0.03 c 1.55±0.07 c - - -
0.5 0.01 48±1 c 51±2 a 41±1 a 2.40±0.03 c 2.55±0.07 a 2.05±0.03 a + - -
0.5 0.01 2.0 62±1 a 50±1 a 42±2 a 3.10±0.03 a 2.50±0.03 a 2.10±0.07 a - - -
0.75 0.01 52±2 b 49±3 a 43±2 a 2.60±0.07 b 2.45±0.1 a 2.15±0.07 a + - -
0.75 0.01 2.0 62±2 a 50±3 a 42±2 a 3.10±0.07 a 2.50±0.1 a 2.10±0.07 a - - -
1.0 0.01 37±2 e 45±4 b 37±2 b 1.85±0.07 e 2.25±0.13 b 1.85±0.07 b + - -
1.0 0.01 2.0 45±2 d 43±3 b 35±2 b 2.25±0.07 d 2.15±0.1 b 1.75±0.07 b - - -

Table 6

The effects of different hormone and hormone concentrations on making robust seedling of 3 Bergenia species"

Concentration (mg·L-1) Average height
before culture (cm)
Average height
after culture (cm)
Adding average
height (cm)
Other condition
6-BA IBA
0.01 0.01 1.1 1.9 0.8±0.1 e Undifferentiated, no root
0.02 0.01 1.2 2.1 0.98±0 e Undifferentiated, no root
0.03 0.01 1.1 2.0 0.98±0 e Undifferentiated, no root
0.04 0.01 1.3 2.5 1.28±0.1 d Undifferentiated, no root
0.05 0.01 1.2 2.1 0.98±0.1 e Differentiated, no root
0.04 0.02 1.1 2.6 1.58±0.1 c Undifferentiated, no root
0.04 0.05 1.2 2.3 1.18±0.1 d Undifferentiated, no root
0.04 0.1 1.1 3.3 2.28±0.1 b Undifferentiated, no root
0.04 0.2 1.3 4.1 2.88±0.2 a Undifferentiated, rootage

Table 7

The effects of different hormone and hormone concentrations on rooting of 3 Bergenia species"

Concentration (mg·L-1) No. of roots (individual) Rooting rate (%) Callus status
NAA IBA VC A B C A B C A B C
0.1 2.0 4±1 f 5±0 d 3±1 e 20±0.05 f 25±0 d 15±0.05 e A small amount of browning A small amount of browning A small amount of browning
0.5 2.0 9±2 d 9±2 c 7±1 d 45±0.1 d 45±0.1 c 35±0.05 d A small amount of browning A small amount of browning A small amount of browning
1.0 2.0 11±2 c 13±2 b 10±1 c 55±0.1 c 65±0.1 b 50±0.05 c A small amount of browning A small amount of browning A small amount of browning
2.0 2.0 12±2 b 11±2 c 12±2 b 55±0.1 c 60±0.1 b 60±0.1 b Severe browning Severe browning Severe browning
0.1 2.0 7±1 e 7±0 cd 7±1 d 35±0.05 e 35±0 cd 35±0.05 d No
browning
No browning No browning
0.5 2.0 17±2 a 18±1 a 16±2 a 85±0.1 a 90±0.05 a 80±0.1 a No
browning
No browning No browning
1.0 2.0 17±2 a 16±3 a 15±2 a 85±0.1 a 80±0.15 a 75±0.1 a No
browning
No browning No browning
2.0 2.0 16±2 b 17±3 a 15±3 a 80±0.1 b 85±0.15 a 75±0.15 a A small amount of browning A small amount of browning A small amount of browning
0.5 0±2 g 17±1 a 15±2 a 0±0.1 g 85±0.05 a 75±0.1 a Browning death Normal Normal

Figure 2

The phenotype of abnormal seedlings and field grown plants of 3 Bergenia species(A), (B) The seedlings of abnormal apex; (C) Tissue culture seedlings were transplanted for half a year; (D) Flowering phenotype of two years old tissue culture plants; (E) Phenotypic changes of leaves of two years old tissue culture plants in winter"

Figure 3

The amplification results of UBC854 primers in 3 Bergenia species(A) The amplification results of UBC854 primers in B. crassi- folia; (B) The amplification results of UBC854 primers in B. scopulosa; (C) The amplification results of UBC854 primers in B. purpurascens; (D) The amplification results of UBC854 primers in 50 generation seedlings of 3 Bergenia species. M: DNA marker; MP: Maternal plant; 20 G: 20 generations; 30 G: 30 generations; 40 G: 40 generations; 50 G: 50 generations; BC: B. crassifolia; BS: B. scopulosa; BP: B. purpurascens"

Figure 4

The comparison of average genetic variation in 3 Bergenia species"

1 董成梅, 杨丽川, 邹澄, 赵沛基, 蒲洪, 张滢 (2012). 岩白菜素的研究进展. 昆明医学院学报 (1), 150-154.
2 丰先红, 李健, 罗孝贵 (2010). 植物组织培养中体细胞无性系变异研究. 中国农学通报 26(14), 70-73.
3 康建坂, 朱海生, 李大忠, 李永平, 汪伟裁, 温庆放 (2010). 应用ISSR技术分析苦瓜种质资源的多态性. 福建农业学报 25, 597-601.
4 李文春, 郭凤根, 张丽梅, 于虹漫, 李信, 林春 (2006). 岩白菜研究现状与展望. 云南农业大学学报 21, 845-850.
5 刘锋, 江涛, 任素梅 (2004). 熊果苷合成研究进展. 日用化学工业 34, 242-244.
6 刘福平 (2010). 植物体细胞无性系变异的遗传基础及主要影响因素. 基因组学与应用生物学 29, 1142-1151.
7 刘敏, 郝秀英, 徐琴, 波拉提, 康喜亮, 王晓军 (2009). 野生花卉厚叶岩白菜组织培养及再生体系的建立. 安徽农业科学 37, 3455-3456, 3462.
8 刘石泉, 李小军, 周根余 (2005). 铁皮石斛不同繁殖代数遗传稳定性RAPD的研究. 江南大学学报(自然科学版) 4, 518-521.
9 刘思泱, 于卓, 蒙美莲, 马艳红, 刘宇杰, 甘霖, 张自强, 李长青 (2010). 6个彩色马铃薯品种的ISSR分析. 华北农学报 25(5), 117-120.
10 吕秀立, 郭小芳, 施季森, 沈烈英 (2016). 厚叶岩白菜叶片离体培养研究. 上海交通大学学报(农业科学版) 34(6), 50-54.
11 吕秀立, 沈烈英, 王健, 王景先 (2013a). 厚叶岩白菜组织培养技术. 林业科技开发 27, 116-118.
12 吕秀立, 王健, 沈烈英, 崔心红, 张春英, 孙伟墨 (2013b). 厚叶岩白菜离体培养和快速繁殖的方法. 中国专利, ZL20121- 0069616.5. 2013-09-18.
13 吕秀立, 张春英, 沈烈英, 关媛, 冯永辉, 施季森 (2017). 岩白菜属植物需求状况及发展趋势分析. 中国农学通报 33(22), 53-57.
14 彭红卫, 赵刚, 权秋梅, 黎云祥, 王潇, 王辉, 高泽梅 (2011). 岩白菜组培中POD活性的研究. 西华师范大学学报(自然科学版) 32, 227-230.
15 田憬若, 谭秀山, 吴婷婷, 孙惜时, 谈甜甜, 任娇 (2014). 岩白菜素的生物活性的研究进展. 食品研究与开发 35(5), 128-132.
16 王碧霞, 赵欢, 黎云祥 (2012). 岩白菜属植物研究的新进展. 光谱实验室 29, 367-370.
17 王继良, 何瑾, 邹澄, 黎其万 (2006). 岩白菜素的研究进展. 中国民族民间医药杂志 (6), 321-325.
18 文晓鹏, 邓秀新 (2003). 利用细胞学和分子标记检测刺梨愈伤组织的遗传稳定性. 果树学报 20, 467-470.
19 杨丽云, 陈翠, 汤王外, 康平德 (2010). 药用植物岩白菜种子发芽特性研究. 种子 29(12), 81-82, 86.
20 袁菊丽, 索建兰 (2011). 岩白菜属药用植物的研究进展. 宝鸡文理学院学报(自然科学版) 31(1), 46-50.
21 曾霞, 庄南生 (2002). 木薯分子标记研究进展. 华南热带农业大学学报 9(1), 6-12.
22 赵桂茹, 王仕玉, 郭凤根, 龙雯虹, 张丽梅, 陈严平, 周平 (2013). 不同光照强度对2年生岩白菜生长的影响. 西部林业科学 42(5), 93-97.
23 赵谦, 杜虹, 庄东红 (2007). ISSR分子标记及其在植物研究中的应用. 分子植物育种 5(6S), 123-129.
24 朱军, 李晓瑾, 贾晓光, 李红侠 (2012). 新疆珍稀药用植物厚叶岩白菜离体快繁研究. 时珍国医国药 23, 2308-2309.
25 Allen GC, Flores-Vergara MA, Krasynanski S, Kumar S, Thompson WF (2006). A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide.Nat Protoc 1, 2320-2325.
26 Reisch BI (1988). Genetic instability in plant cell cultures: utilization in plant breeding and genetic studies. In: Pais MSS, Mavituna F, Novais JM, eds. Plant Cell Biotechnology. Berlin Heidelberg: Springer. pp. 87-95.
27 Zietkiewicz E, Rafalski A, Labuda D (1994). Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification.Genomics 20, 176-183.
No related articles found!
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
[1] Hu Shi-yi. Lipoid Bodies in Plant Tissues[J]. Chin Bull Bot, 1994, 11(04): 49 -51 .
[2] CHENG Hong-Yan. Introduction of State Key Laboratory of Biomembrane and Membrane Biotechnology[J]. Chin Bull Bot, 1998, 15(04): 78 .
[3] Liu Dong-zhuo and Li Lan. The Karyotype Analysis of Solanum pseudocapsicum[J]. Chin Bull Bot, 1992, 9(03): 50 .
[4] WANG Bao-Shan;LI De-Quan;ZHAO Shi-Jie;MENG Qing-Wei and ZOU Qi. Effects of Iso-osmotic NaCl and KCl Stress on Growth and Gas Exchange of Sorghum Seedlings[J]. Chin Bull Bot, 1999, 16(04): 449 -453 .
[5] LI Yao-Dong WEI Yu-Ning XU Ben-Mei. Study on the ABA Content and SOD Activity in Ancient Lotus and Modern Lotus Seeds[J]. Chin Bull Bot, 2000, 17(05): 439 -442 .
[6] LI Zhong-Kui HU Hong-Jun LI Ye-Guang. Advances in Molecular Phylogenetic Relationship of Volvocales[J]. Chin Bull Bot, 2002, 19(04): 419 -424 .
[7] WANG Ting SU Ying-Juan ZHU Jian-Ming HUANG Chao LI Xue-Yan. PCR_RFLP Analysis of rbc L Genes in Taxaceae and Related Taxa[J]. Chin Bull Bot, 2001, 18(06): 714 -721 .
[8] . [J]. Chin Bull Bot, 1994, 11(专辑): 51 .
[9] Hui Liu, Danli Guo, Darun Cai, Xianzhong Huang. Heterologous overexpression of ApZFP Promotes Flowering and Improves Abiotic Tolerance in Arabidopsis thaliana[J]. Chin Bull Bot, 2016, 51(3): 296 -305 .
[10] Dong Shu-ting, Hu Chang-hao, Yue Shou-song, Wang Qun-ying, Gao Rong-qi, Pan Zi-long. The Characteristics of Canopy Photosynthesis of Summer Corn (Zea mays) and its Relation with Canopy Structure and Ecological Conditions[J]. Chin J Plan Ecolo, 1992, 16(4): 372 -378 .